PROJECT 3 SUMMARY Human cytomegalovirus (HCMV) is a ?-herpesvirus infecting 44-100% of the population and remains a significant cause of morbidity and mortality in solid organ transplant (SOT) and allogeneic hematopoietic stem cell transplant (SCT) recipients. Infection in SCT patients is often associated with myelosuppression and graft failure due to virus reactivation from latency, but the associated mechanisms are still largely unknown. HCMV encodes multiple latency-associated gene products including the chemokine receptor US28, which binds CC- chemokines as well as the CX3C-chmokine Fractalkine. US28 signals in both ligand-independent and ligand- dependent manners, but the downstream signaling pathways and consequences are unique for each. Importantly, herein we demonstrate that US28 is required for latency and/or reactivation in both in vitro CD34+ hematopoietic progenitor cells (HPCs) as well as in a humanized mouse model. In addition, we show that US28 is sufficient to promote CD34+ HPC differentiation into myeloid lineage cells. Therefore, based upon our exciting new findings, we hypothesize that US28 ligand-independent signaling during latency helps to maintain latency and that ligand-specific signaling promotes CD34+ HPC differentiation into a reactivation/replication competent myeloid lineage cell to ensure virus replication under appropriate conditions including inflammation. US28 signaling may also contribute to other aspects of latency by acting as a biosensor to promote migration towards inflammatory chemokines, and possibly by increasing immediate early gene expression during reactivation. Defining the role of US28 in latency and reactivation, determining how US28 signaling intersects with EGFR signaling, and understanding how US28 signaling influences other HCMV proteins (Projects 1 and 4) and miRNAs (Project 2) expressed during latency and manipulating EGFR signaling (Project 5) are the goals of this proposal. We propose the following specific aims: 1) To determine how US28 signaling in CD34+ HPCs intersects with EGFR signaling pathways using a multi-omics approach; 2) To determine what molecular characteristics of US28 mediate HCMV latency and reactivation using both in vitro CD34+ HPCs and in vivo humanized BLT mouse models; 3) To determine what US28 signaling pathways promote CD34+ HPC hematopoiesis also using both in vitro CD34+ HPC and in vivo huBLT models. Results of this study will generate new virus latency and reactivation paradigms promoting the development of novel therapies to prevent virus reactivation and to treat HCMV-mediated myelosuppression.